Technical Abstract:
Background: Edwardsiella ictaluri is the etiological agent of enteric septicemia of catfish, which is the most common disease of channel catfish (Ictalurus punctatus) and is responsible for $50 - 60 million economic losses to catfish producers annually in the Southeastern U.S. In the course of studying Ed. ictaluri pathogenesis, we initially immunoscreened the Ed. ictaluri genomic libraries, and tentatively identified one clone, a FK506 binding protein. In this study, we further characterized and expressed this FK506 binding protein.
Method: Genomic DNA of Ed. ictaluri was isolated, and genomic DNA libraries were constructed by using a GenomeWalker Universal kit. The FK506 binding protein gene was amplified by PCR. DNA sequencing and analyzed were carried out according to the standard protocol. The full-length of FK506 binding protein DNA was expressed in Escherichia coli BL21 (DE3) according to the standard protocol, and the recombinant protein was purified by a PrepEase His-tagged Protein Purification kit. The purified protein was further confirmed by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF). The enzyme activity was determined according to the standard protocol.
Results: The complete sequence of the Ed. ictaluri FK506 binding protein gene had 612 nucleotides, potentially encoding a 203-amino-acid peptide with a calculated molecular mass of 20.3 kDa. By comparing with other known FK506 binding proteins deposited in GenBank, the Ed. ictaluri FK506 binding protein had a high degree of homology to those from Enterobacteriaceae, but not from non- Enterobacteriaceae. When the clone was expressed in E. coli BL21 (DE3), this recombinant protein had a molecular weight of about 25.3 kDa determined by SDS-PAGE. The protein was confirmed by MALDI-TOF as an FKBP-type peptidyl-prolyl isomerase. The enzyme activity of the recombinant protein had N-Suc-Ala-Ala-Pro-Phe p-nitroanilide substrate specificity.
Conclusion: Further study of this enzyme may hold important insights in E. ictaluri pathogenesis in catfish.